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A high throughput method for genome-wide analysis of retroviral integration.

Mantovani J, Holic N, Martinez K, Danos O, Perea J - Nucleic Acids Res. (2006)

Bottom Line: Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression.Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences.VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events.

View Article: PubMed Central - PubMed

Affiliation: Genethon-CNRS-Université d'Evry Val d'Essonne, UMR 8115, Evry, France.

ABSTRACT
Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20-25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.

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Evolution of the number of sequenced tags in function of cumulated number of sequenced nucleotides. The 2480 tags generated by running VITA on a HeLa cells were mapped on the human genome and classified in different categories. Curves represent ‘total different tags’ (diamond), ‘unique hits’ (square), ‘no hits’ (triangle) and ‘multiple hits’ (cross) (see Discussion).
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fig3: Evolution of the number of sequenced tags in function of cumulated number of sequenced nucleotides. The 2480 tags generated by running VITA on a HeLa cells were mapped on the human genome and classified in different categories. Curves represent ‘total different tags’ (diamond), ‘unique hits’ (square), ‘no hits’ (triangle) and ‘multiple hits’ (cross) (see Discussion).

Mentions: In order to estimate the complexity of the tag population we represented the number of different tag in each category (‘one hit’, ‘no hit’, ‘multi hit’) as a function of the cumulated number of sequenced nucleotides (Figure 3). All the curves are far from the asymptote, indicating that the tag population can still be extensively exploited.


A high throughput method for genome-wide analysis of retroviral integration.

Mantovani J, Holic N, Martinez K, Danos O, Perea J - Nucleic Acids Res. (2006)

Evolution of the number of sequenced tags in function of cumulated number of sequenced nucleotides. The 2480 tags generated by running VITA on a HeLa cells were mapped on the human genome and classified in different categories. Curves represent ‘total different tags’ (diamond), ‘unique hits’ (square), ‘no hits’ (triangle) and ‘multiple hits’ (cross) (see Discussion).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636494&req=5

fig3: Evolution of the number of sequenced tags in function of cumulated number of sequenced nucleotides. The 2480 tags generated by running VITA on a HeLa cells were mapped on the human genome and classified in different categories. Curves represent ‘total different tags’ (diamond), ‘unique hits’ (square), ‘no hits’ (triangle) and ‘multiple hits’ (cross) (see Discussion).
Mentions: In order to estimate the complexity of the tag population we represented the number of different tag in each category (‘one hit’, ‘no hit’, ‘multi hit’) as a function of the cumulated number of sequenced nucleotides (Figure 3). All the curves are far from the asymptote, indicating that the tag population can still be extensively exploited.

Bottom Line: Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression.Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences.VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events.

View Article: PubMed Central - PubMed

Affiliation: Genethon-CNRS-Université d'Evry Val d'Essonne, UMR 8115, Evry, France.

ABSTRACT
Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20-25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.

Show MeSH
Related in: MedlinePlus